The present invention relates to a class of molecules specified as novel multipurpose antibody derivatives. The invention further relates in particular to such antibody derivatives that have two or more antigen binding parts, derivatives that have at least two antigen binding parts, combined with at least one other function, such as a toxin, an enzyme, a cytokine, a hormone or a signalling molecule, and derivatives that have an antigen binding part, combined with at least two other functions.
Due to their versatility, multipurpose antibody derivatives (mpAbs), such as bispecific antibodies (BsAb), immunotoxins and bifunctional antibodies are promising tools in the treatment of various (human) diseases. The first arm usually allows to specifically recognize a target cell (e.g. cancer cell) by means of an antigen binding function, while another determinant may be directed through an antigen binding function towards a second cell type (e.g. a cytotoxic T cell), or it may be a toxin, an enzyme (e.g. to locally cleave and activate a prodrug), a cytokine, a hormone or a signalling molecule.
The difficulty of producing functional BsAb in sufficient quantity and purity is still hampering the more general use of BsAb in clinical applications. When using the guadroma technology only 10% of the immunoglobulin pool is the correct, bispecific antibody. Therefore, time consuming and costly purification procedures are inevitable.
Chemical reassociation of antibody fragments suffers from loss of affinity by protein denaturation or unorthodox disulphide bond formation, as well as from the use of a chemical cross-linker, generating inactive, chemically modified structures.
Both these classical methods producing BsAb give rather low yields. Recombinant DNA methodology and antibody engineering has greatly facilitated the production of antibody derivatives in heterologous expression systems. By genetic fusion of various antibody fragments to generate BsAb, the normal tetrameric antibody structure (H+L)2 is reduced. When the total Fc-portion is included, the self-association of the disulphide bridges in the hinge region reduces considerably the yield of heterodimeric BsAb. Hence purification away from bivalent, homodimeric by-products is still required. In order to improve the level of heterodimeric, bispecific product, a xe2x80x9cKnobs-into-holesxe2x80x9d principle has been developed to engineer the CH3 domains in the Fc-tail for preferential heterodimerization. The molecule proposed by Ridgway et al. (1996) comprises a complete Fc portion, which increases the molecular weight of the final protein beyond the optimal size for biodistribution. Furthermore, the Fc portion can interact with a multitude of Fc receptors present on various cells in the body, which can deviate the binding of this molecule to aspecific targets.
Small antibody-derivatives (such as sFv, bssFv, (diabodies) have the advantage of easy penetration in solid tumors; moreover, partly because of the absence of high disulphide containing hinge regions, they can be produced in high amounts in heterologous expression systems. However, due to their small size, these molecules are generally cleared too rapidly from the circulation to allow efficient accumulation at the tumor site, while molecules of intermediate size have improved serum stability and retain satisfactory tissue penetration.
In order to achieve medium sized heterodimers, sFv have been linked by incorporating an additional peptide, leucine zippers, amphiphatic helices or streptavidin. These heterodimerization extensions, however, might be immunogenic.
Similar problems are encountered in the preparation of immunotoxins and antibody derivatives having an enzymatic function. Monovalent single chain Fv fragments (sFv) or disulfide stabilized Fv fragments (dsFv) are predominantly used to construct toxin fusions. This results in weaker binding and poor internalization due to the monovalent binding, and rapid blood clearance due to the small molecule size.
In view of the above it is the object of the present invention to provide a class of molecules, specified as novel multipurpose antibody derivatives that can be efficiently prepared without many by-products, that have an intermediate size and that combine two or more antigenic binding sites, or one antigenic binding site with two or more other functions in one molecule.
This is achieved according to the invention by multipurpose antibody derivative, comprising the CL and VL domains of a first antibody with a desired first antigen binding specificity, the CH1 and VH domains of the said first antibody interacting with the CL and VL domains, and one or more other molecules having at least one further purpose coupled to one or more of the domains of the first antibody.
The invention is based on the potential of the specific VL-CL:VH-CH1 (referred to as xe2x80x9cL:Fdxe2x80x9d) interaction to drive disulphide-stabilized heterodimerization of recombinant antibody-derived fusion proteins. The use of the L:Fd interaction which can be both natural or chimeric to drive heterodimerization has several advantages. First of all, their natural heterodimeric interaction circumvents the need for protein engineering to achieve complementarity. Furthermore, the interaction is very strong, in contrast to L:L homodiners which are only poorly formed or Fd:Fd homodimers which were never detected in eukaryotic expression systems. Also, in bacterial expression systems the Fd chain alone is aberrantly folded (Ward, 1992). Finally, a single, natural disulphide bridge stabilizes the L:Fd heterodimer.
Each of the two domains of the light and heavy chain can be extended with another molecule (e.g. VL or VH region, a sFv, a toxin, an enzyme such as a prodrug cleaving enzyme, a cytokine, a hormone, a signalling molecule, etc.).
Thus, the invention relates to a class of molecules specified herein as novel xe2x80x9cmultipurpose antibody derivativesxe2x80x9d. This class of molecules is created by heterodimerization of two constituting components. Heterodimerization is obtained by the specific heterotypic interaction of a chosen CH1-VH combination of immunoglobulin domains, with a chosen CL-VL combination of immunoglobulin domains. The VHCH1-VLCL interaction is proposed as a very efficient heterodimerization scaffold that could be efficiently produced. By choosing the tappropriate VH and VL domains in the VHCH1 and VLCL context, a binding specificity can be constituted by the heterodimerization scaffold itself. One or both of the comprising VHCH1 and VLCL chains can thus be extended at either the N- or the C-terminus or both with other molecules, such as a toxin, an enzyme, a cytokine, a hormone or a signalling molecule and derivatives that have an antigen binding part for the purpose of combining these molecules with each other.
The construction of the Fab part of the antibody, fixed to relatively simple molecules such as bacterial alkaline phosphatase, or a truncated mutant form of Pseudomonas exotoxin has been described before (Ducancel et al., 1993, Choe et al., 1994). However, unexpectedly it was found according to the invention that the L:Fd interaction is still able to drive the heterodimerization when one of the chains of the Fab is fused to a complex molecule as a single-chain antibody fragment. Even more unexpectedly, it was found that also both chains of the Fab may be fused to other molecules, without affecting the ability of the molecules to form preferentially heterodimers.
ScFv molecules consist of domains (VL and VH) of the same nature as can be found in the Fd and L chains, so wrongly formed non-functional derivatives could easily be expected. However, the findings as illustrated in the examples unexpectedly show that such molecules can be produced efficiently and is proven functional for all its components.
Surprisingly, this could be achieved with peptide linkers as short as a few amino acids. By excluding the hinge-region, dimerization of the Fab-scFv fusion is omitted. Homodimerization of some specificities might induce unwanted activating or inhibiting functions with effector cells. In order to avoid this, homodimerization through e.g. the hinge region can be avoided by excluding this region in the Fab-scFv molecule.
The other molecule(s) can be fused either to the C-terminus of the CH1 the N-terminus of the VH, the C-terminus of the CL and/or the N-terminus of the VL. In total, the invention offers 15 different variant types of combinations of other molecules with the L+Fd construct as a scaffold. The variant types are summarized in table 1. Each variant type can in turn be provided with various kinds of other molecules.
The L:Fd acts as a xe2x80x9ccarrierxe2x80x9d for the other molecule. In the case of an sFv as the other molecule, the total size of the sFv is increased due to the presence of the carrier. As a consequence it will not have the disadvantage of known sFv""s or bssFv that are cleared too rapidly from the circulation. The L and Fd chains can if desired, constitute a binding specificity of their own. In this case, the L and Fd chains contribute a function of their own, apart from serving as a heterodimerization signal.
When a molecule of the invention combines two (different of equal) functions, it is called bifunctional. Similarly, when a molecule of the invention combines three or more than three different or equal functions, it is called trifunctional, respectively multifunctional. When a molecule of the invention is combining two, three or more antibody parts having a different specificity, it is called bi-, respectively tri- or multispecific. When a molecule of the invention is combining two, three or more antibody parts having the same specificity, it is called bi-, respectively, tri- or multivalent for the binding specificity.
In a first preferred embodiment, the invention provides for a novel, recombinant mpAB that is a bispecific, bifunctional antibody (BsAb) when the specificities are different or bivalent, bifunctional antibody (BvAb) when the specificities are the same. These are based on the fusion of a Fab and a sFv, which is fused to the C-terminus of CH1 or CL. This molecule will have an intermediate size of about 80 kDa, satisfies the aforementioned criteria and incorporates preferential heterodimerization through its L:Fd domains.
According to a second preferred embodiment a similar antibody is provided which is also based on the fusion of a Fab and a sFv, but in this case the latter is fused to the N-terminus of VH or the N-terminus of VL. In a third preferred embodiment, the invention provides for a novel, recombinant bispecific, trifunctional or bivalent, trifunctional mpAB that is an immunotoxin based on the fusion of a BsAb or a BvAb according to the first embodiment and a toxin, which is fused to the C-terminus of the heavy chain of the Fab that does not carry the sFv.
According to a fourth preferred embodiment, the invention provides for a novel, recombinant bispecific, trifunctional or bivalent, trifunctional mpAB that is called a catalytic antibody (cAb) based on the fusion of a BsAb or a BvAb and an enzyme, which is fused to the C-terminus of the heavy chain of the Fab that does not carry the sFv.
According to a fifth preferred embodiment, the invention provides for a novel, recombinant bispecific, trifunctional or bivalent, trifunctional mpAB that is combined with a hormone, a cytokine or a signalling function by fusing of a molecule with said activity to an BsAb or a BvAb according to the first embodiment.
According to a sixth preferred embodiment both the C-terminus of CH1 and the C-terminus of CL are fused to a sFv, resulting in a molecule with three antigen binding parts. This molecule is trifunctional, and can be trivalent monospecific, bivalent bispecific or monovalent trispecific.
Thus, this invention offers inter alia the possibility to create bivalent trifunctional immunotoxins (i.e. molecules that are intended for two purposes, namely bivalent antigen binding and toxicity) or trispecific (i.e. three antigen specificities), antibodies. In the latter case not only the CH1, but also the CL is extended with an sFv.
The other molecule can be linked to the L or Fd antibody part(s) directly or via a linker. The presence of a linker of at least 1, preferably more than 3 amino acids can be used to avoid steric hindrance between two or more antigen binding sites and between antigen binding site(s) and the active center of the other molecule. Linkers other than amino acid chains may also be used.
According to one specifically preferred embodiment of the invention various anti murine CD3xcex5-single-chain fragments (sFv) were fused to the C-terminus of CH1 of an Fd fragment specific for human placental alkaline phosphatase (hPLAP). This Fab-sFv bispecific antibody derivative (of the general formula Fab-linker-sFv, wherein the linker is e.g. EPSG but can be variable in sequence and length) can be used to link cytotoxic cells to tumor cells.
The fusion product was further improved for reaching far apart antigens by providing a sufficiently long spacer sequence (of the general formula Fab-linker-sFv, wherein the linker is e.g. EPSGP(G4S)3M but can be variable in sequence and length). After eukaryotic secretion, specific heterodimerization between the corresponding anti-hPLAP light chain and the Fd fragment occurred, where the latter carried a functional sFv. Upon expression in mammalian cells more than 90% of the immunoglobulin material in the medium was the specific heterodimer, with only minor contamination of light chain derived homodimers and monomers, which did not show hPLAP binding capacity. Homodimers from the heavy chain derived VH-CH1 fused to the anti CD3xcex5 sFv were never observed.
The Fab-sFv fusion protein between the anti murine CD3xcex5 sFv and the anti-hPLAP-Fab here described is an example for the efficient production of specific, disulphide stabilized heterodimers which can be used for making bispecific antibodies. The invention is not limited to this particular example. Other antigen binding specificities can be used and for the other purpose or function there is also a variety of options. The invention lies in principle in the finding that the L:Fd interaction is highly specific and can be used as a heterodimeric scaffold to construct a new type of mpAb. The VL and CL domains in the L chain, as well as the Vh and CH1 domains in the Fd chain do not necessarily have to be derived from the same antibody.
The derivatives of the invention can, be used in the treatment of tumors, in the treatment of various infected cells, in the treatment of autoimmune diseases or thrombosis. Moreover the derivatives of the invention can be used to direct a virus towards immunological effector cells, to induce or resolve blood clotting, to eliminate specific cell types in vitro or in vivo, to establish or improve transfections, or in diagnosis.
The invention further relates to DNA constructs encoding the heavy chain domains of an antibody derivative of the invention, comprising suitable transcription and translation regulatory sequences operably linked to sequences encoding the VH and CH1 domains of the first antibody and optionally a coding sequence for the other molecule operably linked thereto.
In such a DNA construct the coding sequence for the other molecule may consist of DNA sequences encoding the VL and VH domains of a second antibody, which DNA sequences are operably linked to each other in either one of the sequences 5xe2x80x2-VL2-VH2-3xe2x80x2 or 5xe2x80x2-VH2-VL2-3xe2x80x2.
In the DNA construct a DNA sequence encoding a linker sequence may be incorporated between one or more of the VH, CH1, VL2 and/or VH2 coding sequences and/or the coding sequence for the other molecule. The linker helps in avoiding steric hindrance between the various domains.
A particularly preferred DNA construct, designated as pCA2C11sFvE6Hf, is obtainable from E. coli DH5xcex1 cells deposited on Oct. 15, 1997 at the Belgian Coordinated Collection of Microorganisms and given the deposit accession no. LMBP3715. Another preferred DNA construct is designated as pCAE6HfGS2C11sFv (also identified as pCAE6H2sc2C11H) and obtainable from E. coli MC1061 cells deposited on Oct. 15, 1997 at the Belgian Coordinated Collection of Microorganisms and given the deposit accession no. LMBP3716.
Furthermore the invention relates to DNA construct encoding the light chain domains of an antibody derivative of the invention, comprising suitable transcription and translation regulatory sequences operably linked to sequences encoding the VL and CL domains of the first antibody and optionally a coding sequence for the other molecule operably linked thereto. The coding sequence for the other molecule may consist of DNA sequences encoding the VL and VH domains of a second antibody, which DNA sequences are operably linked to each other in either one of the sequences 5xe2x80x2-VL2-VH2-3xe2x80x2 or 5xe2x80x2-VH2-VL2-3xe2x80x2.
Also in this DNA construct a linker sequence can be incorporated between one or more of the VL, CL, VL2 and/or VH2 coding sequences and/or the coding sequence for the other molecule.
According to a further aspect the invention relates to a set of DNA constructs for producing multipurpose antibody derivatives of the invention, comprising any one of the constructs described above together with a construct encoding at least the light domains VL and CL of the first antibody or together with a construct encoding at least the heavy domains VH and CH of the first antibody, depending on whether the other construct encodes the heavy or light domains of the first antibody.
In a first embodiment the set consists of vector pCAE6H2sc2C11H and vector pCAG6SE6L. In an alternative embodiment the set consists of vector pCA2C11sFvE6Hf and vector pCAG6SE6L. Those sets can be used for producing multipurpose antibody derivatives of the invention in heterologous expression host cells. The invention also relates to a method for producing multipurpose antibody derivatives, comprising expression of such a set in heterologous expression host cells. The host cells may be E. coli cells, other bacterial cells, such as Bacillus spp., Lactobacillus spp. or Lactococcus spp.; actinomycetes; yeasts; filamentous fungi; mammalian cells, such as COS-1 cells, HEK cells, insect cells, transgenic animals or plants.
Another aspect of the invention relates to a medical preparation, comprising multipurpose antibody derivatives.
A further aspect of the invention relates to the use of multipurpose antibody derivatives in diagnosis.
According to a final aspect the invention relates to the use of multipurpose antibody derivatives for the preparation of a medicament for the treatment of cancer, infections, parasites, autoimmune diseases, thrombosis.
The term xe2x80x9cpurposexe2x80x9d is used herein to indicate a certain activity or other function, preferably antigen binding specificity, toxicity, signalling or enzymatic activity.
The term xe2x80x9cderivativexe2x80x9d is used herein to refer to molecules other than the classic antibodies consisting of two light chains and two heavy chains, which heavy chains in turn comprise multiple constant domains. The derivatives comprise at least one VL domain, one CL domain, one VH domain and one CH domain.
Derivatives of the present invention can thus be prepared by genetic engineering using methods well known in the art. In the examples that follow, it is described how by genetic engineering, a new type of bispecific antibody with potential use in immunotherapy by redirected cellular cytotoxicity was designed. The design of the antibody was bas d on the very effective and selective heterodimerization of the two antibody-chains, L and Fd. Both the Fd and the L chain can be extended with new determinants, herein called xe2x80x9cother moleculesxe2x80x9d (peptides, domains), either at their N-terminus or C-terminus or both. As an example the molecule Fab (L+Fd) is described extended either at the N-terminus or at the C-terminus of the Fd fragment with a single chain antibody fragment (sFv). The latter, Fab-(G4S)3-sFv, was characterized in detail. (G4S)3 is short for EPSGPGGGGSGGGGSGGGGSM (SEQ ID NO:30). The bispecific species was the predominant product in a heterologous expression system. It had an intermediate molecular weight which is beneficial for serum stability, biodistribution and solid tissue penetration.
The following examples provide the teaching starting from which variants can be prepared. The examples are therefore in no way intended to be limiting the invention. In the examples xe2x80x9cVHxe2x80x9d, xe2x80x9cCH1xe2x80x9d, xe2x80x9cCLxe2x80x9d and xe2x80x9cVLxe2x80x9d are used for domains derived from the first antibody. xe2x80x9cVH2xe2x80x9d and xe2x80x9cVL2xe2x80x9d are used for domains derived from the second antibody. xe2x80x9cVH3xe2x80x9d and xe2x80x9cVL3xe2x80x9d are used for domains derived from the third antibody.